Investigation on the Potential Functions of ZmEPF/EPFL Family Members in Response to Abiotic Stress in Maize

Maize is an important crop used for food, feed, and fuel. Abiotic stress is an important factor affecting maize yield. The EPF/EPFL gene family encodes class-specific secretory proteins that play an important role in the response to abiotic stress in plants. In order to explore and utilize the EPF/EPFL family in maize, the family members were systematically identified, and their chromosomal localization, physicochemical properties, cis-acting element prediction in promoters, phylogenetic tree construction, and expression pattern analysis were carried out using bioinformatics techniques. A total of 18 ZmEPF/EPFL proteins were identified in maize, which are mostly alkaline and a small portion acidic. Subcellular localization results showed that ZmEPF6, ZmEPF12, and ZmEPFL2 are localized in the nucleus and cytoplasm. Analysis of cis-acting elements revealed that members of the ZmEPF/EPFL family contain regulatory elements such as light response, anoxic, low temperature, and hormone response regulatory elements. RT-qPCR results showed that these family members are indeed responding to cold stress and hormone treatments. These results of this study provide a theoretical basis for improving the abiotic stress resistance of maize in future research.


Introduction
Agricultural ecosystems have been adversely affected by abiotic stress, such as high temperature, low temperature, drought, salt, and so on [1].Stomata is one of the key factors for plants to resist adversity stresses, whose size and density change with the environment [2].By altering stomatal density, pore size, and switching time, carbon dioxide uptake in plant photosynthesis can be optimized, and external abiotic stresses can be resisted [3].A large number of ROS accumulated in guard cells promote stomatal closure through activation of calcium channels in the plasma membrane in response to a variety of abiotic stresses [4].Plants could increase drought tolerance by regulating stomatal closure and reducing water transpiration [5,6].
The fossil record indicates that terrestrial plants had stomatal-like structures on their surface over 400 million years ago [7].This feature emerged independently in newly evolved terrestrial plant strains [8].Stomata are composed of guard cells and the pores formed around them, which exist on the surface of most terrestrial plants and act as microscopic pressure-driven valves [7].Each stoma is surrounded by two highly specialized cells called Guard Cells, which require stereotypical asymmetric and symmetric cell division [9].Its cell development process strictly follows the cell division program, ensuring that there is at least one interval of cell separation between each mature stoma [10].
In order to facilitate the correct development of stomata and the absence of intercellular separation between stomata, some extracellular and plasma membrane-bound proteins are essential for the coordinated development of stomata and signaling between Epidermal Pavement Cells [11].These signals include EPF (EPIDERMAL PATTERNING FACTOR) and EPFL (EPIDERMAL PATTERNING FACTOR-Like) signal peptides, leucinerich repetitive ER (ERECTA) series membrane receptor kinases ERECTA/ERECTA-LIKE1 (ERL1), and Leu-rich repetitive membrane protein TMM (Too Many Mouths) [10].During stomatal development, ER family proteins regulate the direction of differentiation of MMC (Meristemoid Mother Cells) and are regulated by EPF [12].
So far, members of the EPF/EPFL family have been identified in several species, such as moss (Physcomitrium patens), barley (Hordeum vulgare), rice (Oryza sativa), wheat (Triticum aestivum L.), and Arabidopsis thaliana [13][14][15][16][17].For example, moss has conserved homologs of EPF, which function together to facilitate the correct development of stomata [13].Plants develop stomata in a manner that employs multiple transcription factors and EPF ligands to more precisely control subsequent differentiation [18].In barley, overexpression of HvEPF1 restricts the access of HvEPF1 to the stomatal developmental pathway and significantly enhances water use efficiency, drought tolerance, and soil water retention performance [14].In rice, overexpression of OsEPF1 and OsEPF2 could reduce the stomatal density and stomatal conductance in leaves, which led to improved water use efficiency [15,19].Moreover, several studies have demonstrated that EPF/EPFL family members are involved in the stomatal development process in Arabidopsis thaliana.EPF1 encodes a small secretory protein that is expressed in stomatal precursor cells and controls stomatal development by regulating asymmetric cell division in the MMC [10].EPF2 regulates the stomatal density of mature leaves and controls the number of cells entering the stomata during leaf development [20].Elevated expression of EPFL9 increases stomatal density and aggregation [21].However, the control of stomatal aggregation by EPFL9 and EPF1, as well as the regulation of stomatal density by EPFL9 and EPF2, are independent of each other [22].
Besides involvement in stomatal regulation, EPFL members are also involved in plant growth, fertility, and yield regulation.For instance, OsEPFL1 has the function of elongating awns in rice [23].OsEPFL2 is involved in the development of awns and large grains, regulating grain length and grain weight [24].Furthermore, OsEPFL6, OsEPFL7, OsEPFL8, and OsEPFL9 negatively regulate the number of spikelets per spike.The osepfl6 osepfl7 osepfl9 triple mutants significantly increased grain yield without affecting spikelet fertility, and specific inhibition of the OsEPFL6-OsER1, OsEPFL7-OsER1, and OsEPFL9-OsER1 ligand receptors could optimize rice panicle architecture [25].
Although the number of EPF/EPFL homologs has been identified in several species, there is still a lack of systematic identification and related research reports on the members of the EPF/EPFL family in maize.In this paper, we conducted a series of bioinformatics analyses to systematically identify and analyze the members of the EPF/EPFL gene family in maize.These results contribute to a better understanding of the evolutionary history and functional mechanisms of the EPF/EPFL family in maize.

Gene Structure Analysis and Protein-Conserved Motif and Domain Prediction
To identify the EPF/EPFL family members in maize, we downloaded the maize genome DNA, cDNA, protein sequence, and maize annotation files from the Ensembl Plants website and performed the protein blast using the Arabidopsis EPF/EPFL family members.A total of 18 EPF/EPFL members were identified in maize, named ZmEPF1-15 and ZmEPFL1-L3, respectively (Figure 1).To visualize the gene structure of maize EPF/EPFL family members, we used TBtools-II (V2.042) software to draw a schematic diagram of gene structure.All 17 maize EPF/EPFL genes contain one or two introns, except for ZmEPF7, which is an intronless gene (Figure 1A).gene structure.All 17 maize EPF/EPFL genes contain one or two introns, except for ZmEPF7, which is an intronless gene (Figure 1A).Next, a prediction of the protein-conserved domain and motif of all maize EPF/EPFL family members was performed.Results of conserved motif prediction showed that there are seven motifs in the maize EPF/EPFL family members, from motif 1 to 7 (Figure 1B).Three family members have one motif, seven members contain two motifs, three members have three motifs, and four members have four motifs (Figure 1A).Prediction of domain showed that there are three conserved domains in the maize EPF/EPFL family members, including the EPF domain, the stomagen-like superfamily domain, and the PLN03207 domain (Figure S1).Among the maize EPF/EPFL family members, 12 members contain EPF domains.ZmEPFL1 contains a stomagen-like superfamily domain, and ZmEPFL2 and L3 contain PLN03207 domains (Figure S1).

Phylogenetic Relationship Analysis
To analyze the phylogenetic relationships of the ZmEPF/EPFL family, phylogenetic tree construction of EPF/EPFL proteins was completed in the three species of Arabidopsis thaliana, Oryza sativa, and Zea mays (including 11 Arabidopsis proteins, 12 rice proteins, and 18 maize proteins) using the MEGA 7 (V7.0.26) software (Figure 2).Six subfamilies were classified, from Class I to Class VI subfamilies.There are five members in the Class I subfamily, five in Class II, two in Class III, three in Class IV, three in Class V, and zero in Class VI, respectively (Figure 2).Next, a prediction of the protein-conserved domain and motif of all maize EPF/EPFL family members was performed.Results of conserved motif prediction showed that there are seven motifs in the maize EPF/EPFL family members, from motif 1 to 7 (Figure 1B).Three family members have one motif, seven members contain two motifs, three members have three motifs, and four members have four motifs (Figure 1A).Prediction of domain showed that there are three conserved domains in the maize EPF/EPFL family members, including the EPF domain, the stomagen-like superfamily domain, and the PLN03207 domain (Figure S1).Among the maize EPF/EPFL family members, 12 members contain EPF domains.ZmEPFL1 contains a stomagen-like superfamily domain, and ZmEPFL2 and L3 contain PLN03207 domains (Figure S1).

Phylogenetic Relationship Analysis
To analyze the phylogenetic relationships of the ZmEPF/EPFL family, phylogenetic tree construction of EPF/EPFL proteins was completed in the three species of Arabidopsis thaliana, Oryza sativa, and Zea mays (including 11 Arabidopsis proteins, 12 rice proteins, and 18 maize proteins) using the MEGA 7 (V7.0.26) software (Figure 2).Six subfamilies were classified, from Class I to Class VI subfamilies.There are five members in the Class I subfamily, five in Class II, two in Class III, three in Class IV, three in Class V, and zero in Class VI, respectively (Figure 2).In addition, we performed the collinearity analysis of EPFs and EPFLs in different species (Arabidopsis thaliana, Zea mays, and Oryza sativa) (Figures S2 and S3).Results showed that there are three homologous genes between Arabidopsis thaliana and Zea mays, while there are 12 homologous genes between Zea mays and Oryza sativa (Figure S2).In Zea mays, there are five homologous pairs of EPF/EPFL genes, including ZmEPF2 and 6, ZmEPF4 and 5, ZmEPF7 and 12, ZmEPF9 and 11, ZmEPFL2 and L3 (Figure S3).These results suggested that maize EPF/EPFL family members are more closely related to rice compared with Arabidopsis, consisting of the phylogenetic tree.

Physicochemical Property Prediction
To get information on the physicochemical properties of ZmEPF/EPFL family members, a comprehensive analysis of the ZmEPF/EPFL family was performed, including subcellular localization, protein length, molecular mass, theoretical isoelectric point (pI), instability index, and grand average of hydropathicity (GRAVY) (Table 1).As shown in Table 1, the relative protein length of the ZmEPF/EPFL members ranges from 113 to 216 amino acids, the relative molecular weight is from 12,115.97 to 23,070.52Da, and the pI is between 6.81 and 10.7.Only one family member has an isoelectric point less than 7, and the rest have isoelectric points greater than 7.The instability indexes (II) were computed to range from 46.20 to 76.84, indicating that all family members may be unstable.The GRAVY ranges from −0.032 to −0.680, indicating that all the family members may be hydrophilic proteins.Subcellular localization prediction revealed that ZmEPF/EPFL family members may localize in the nucleus, plasma membrane, chloroplast, or cytoplasm (Table 1).To further study the characteristics of these proteins, we fused them with pGWB6-ZmEPF6, ZmEPF12, and ZmEPFL2, transformed tobacco leaf epidermis through Agrobacterium-mediated methods, and observed fluorescence signals using fluorescence confocal microscopy, respectively.Results showed that the green fluorescence signals of pGWB6-ZmEPF6, 12, and L2 were observed in the nucleus, cytoplasm, cell wall, or cell membrane (Figure 3), partially consistent with the results of subcellular localization prediction (ZmEPF6: chloroplast, nucleus, and cytoplasm; ZmEPF12: nucleus; ZmEPFL2: chloroplast, nucleus, and cytoplasm).Regarding whether ZmEPF6 and L2 have chloroplast localization signals, this needs to be further investigated by constructing vectors in which GFP is fused to the C-terminus of the target proteins.

Identification and Chromosome Distribution
Based on genome annotation information, we drew the chromosome distribution map of 18 maize EPF/EPFL family members using the TBtools-II software.Results showed

Identification and Chromosome Distribution
Based on genome annotation information, we drew the chromosome distribution map of 18 maize EPF/EPFL family members using the TBtools-II software.Results showed that all 18 ZmEPF/EPFL members were distributed on nine chromosomes randomly from chromosome 1 to 10, except for 7 (Figure 4).that all 18 ZmEPF/EPFL members were distributed on nine chromosomes randomly from chromosome 1 to 10, except for 7 (Figure 4).

Analysis of cis-Acting Elements in Promoters
In order to understand which factors may be related to ZmEPF/EPFL family members, we performed a cis-acting element analysis of the promoter region of ZmEPF/EPFL family members.Results showed that the promoters of the family members contain nineteen cis-element (Figure 5), including abscisic acid responsive element (ten members), anoxic specific inducible element (ten members), defense and stress responsive element (six members), endosperm expression regulatory element (five members), gibberellin responsive element (nine members), light responsive regulatory element (eighteen members), low temperature responsive element (five members), MeJA responsive regulatory element (eighteen members), meristem expression regulatory element (eleven members), auxin responsive regulatory element (three members), drought inducible element (seven members), Light responsive element (thirteen members), salicylic acid responsive element (eight members), anaerobic inducible regulatory element (nine members), auxin responsive element (eight members), zein metabolism regulatory element (eight members), ATrich DNA binding protein binding element (one member), seed specific regulatory element (four members), and cell cycle regulatory element (one member).

Analysis of cis-Acting Elements in Promoters
In order to understand which factors may be related to ZmEPF/EPFL family members, we performed a cis-acting element analysis of the promoter region of ZmEPF/EPFL family members.Results showed that the promoters of the family members contain nineteen cis-element (Figure 5), including abscisic acid responsive element (ten members), anoxic specific inducible element (ten members), defense and stress responsive element (six members), endosperm expression regulatory element (five members), gibberellin responsive element (nine members), light responsive regulatory element (eighteen members), low temperature responsive element (five members), MeJA responsive regulatory element (eighteen members), meristem expression regulatory element (eleven members), auxin responsive regulatory element (three members), drought inducible element (seven members), Light responsive element (thirteen members), salicylic acid responsive element (eight members), anaerobic inducible regulatory element (nine members), auxin responsive element (eight members), zein metabolism regulatory element (eight members), AT-rich DNA binding protein binding element (one member), seed specific regulatory element (four members), and cell cycle regulatory element (one member).that all 18 ZmEPF/EPFL members were distributed on nine chromosomes randomly from chromosome 1 to 10, except for 7 (Figure 4).

Analysis of cis-Acting Elements in Promoters
In order to understand which factors may be related to ZmEPF/EPFL family members, we performed a cis-acting element analysis of the promoter region of ZmEPF/EPFL family members.Results showed that the promoters of the family members contain nineteen cis-element (Figure 5), including abscisic acid responsive element (ten members), anoxic specific inducible element (ten members), defense and stress responsive element (six members), endosperm expression regulatory element (five members), gibberellin responsive element (nine members), light responsive regulatory element (eighteen members), low temperature responsive element (five members), MeJA responsive regulatory element (eighteen members), meristem expression regulatory element (eleven members), auxin responsive regulatory element (three members), drought inducible element (seven members), Light responsive element (thirteen members), salicylic acid responsive element (eight members), anaerobic inducible regulatory element (nine members), auxin responsive element (eight members), zein metabolism regulatory element (eight members), ATrich DNA binding protein binding element (one member), seed specific regulatory element (four members), and cell cycle regulatory element (one member).

Expression Pattern Analysis
To investigate the expression pattern of ZmEPF/EPFL family members, a spatiotemporal expression heatmap was established based on the transcriptome data (Figure 6).As shown in Figure 6, ZmEPFL1 exhibits high expression levels in embryos (24 DAP), ZmEPF3, 9, 11, and L2 exhibit high expression level in leaf zone 2 (stomatal) comparing with other members.ZmEPF2, 7, 12, and L3 exhibit relatively moderate expression levels in internode 7-8, leaf zone 2 (stomatal), and vegetative meristem (16-19 days).ZmEPF15 exhibits a medium expression level in embryos (twenty-four DAP) and whole seeds (four DAP).

Expression Pattern Analysis
To investigate the expression pattern of family members, a spatiotemporal expression heatmap was established based on the transcriptome data (Figure 6).As shown in Figure 6, ZmEPFL1 exhibits high expression levels in embryos (24 DAP), ZmEPF3, 9, 11, and L2 exhibit high expression level in leaf zone 2 (stomatal) comparing with other members.ZmEPF2, 7, 12, and L3 exhibit relatively moderate expression levels in internode 7-8, leaf zone 2 (stomatal), and vegetative meristem (16-19 days).ZmEPF15 exhibits a medium expression level in embryos (twenty-four DAP) and whole seeds (four DAP).

Expression Levels of ZmEPF/EPFL Genes in Response to Abiotic Stresses
To investigate the potential response to abiotic stresses of ZmEPF/EPFL family members, expression heatmaps under different abiotic stresses were established, including cold stress, heat stress, salt stress, UV stress, and water deficit stress (Figure 7).Results of heatmaps showed that expression of six members increased under cold stress, including ZmEPF3, 5, 8, 13, 15, and L1.The expression of two members (ZmEPF13 and L1) increased under heat stress.Under salt stress, only two members (ZmEPF11 and 12) showed increased expression.Under UV stress, seven members exhibited increased expression levels, including ZmEPF1, 2, 10, 11, 12, 14, and 15 (Figure 7A).
Furthemore, the transcription level of ZmEPF/EPFL family members was also analyzed under different degrees of water deficit (Figure 7B).There were no significant

Expression Levels of ZmEPF/EPFL Genes in Response to Abiotic Stresses
To investigate the potential response to abiotic stresses of ZmEPF/EPFL family members, expression heatmaps under different abiotic stresses were established, including cold stress, heat stress, salt stress, UV stress, and water deficit stress (Figure 7).Results of heatmaps showed that expression of six members increased under cold stress, including ZmEPF3, 5, 8, 13, 15, and L1.The expression of two members (ZmEPF13 and L1) increased under heat stress.Under salt stress, only two members (ZmEPF11 and 12) showed increased expression.Under UV stress, seven members exhibited increased expression levels, including ZmEPF1, 2, 10, 11, 12, 14, and 15 (Figure 7A).
Furthemore, the transcription level of ZmEPF/EPFL family members was also analyzed under different degrees of water deficit (Figure 7B).There were no significant differences in the expression of the four family members in the presence or absence of a water deficit, including ZmEPF3, 13, 15, and L1.After 6 h under mild water deficit stress, the transcription levels of five members increased, including ZmEPF7, 8, 10, 12, and L3.After 24 h under mild water deficit stress, the transcription levels of seven members increased, including ZmEPF6, 7, 9, 11, 12, L2, and L3.Compared with a mild water deficit, more family members responded to a severe water deficit.After 6 h under severe water deficit stress, the transcription levels of seven members increased, including ZmEPF2, 7, 8, 10, 12, L2, and L3.After 24 h under severe water deficit stress, the transcription levels of ten members increased, including ZmEPF2, 6, 7, 8, 9, 10, 11, 12, L2, and L3.
differences in the expression of the four family members in the presence or absence of a water deficit, including ZmEPF3, 13, 15, and L1.After 6 h under mild water deficit stress, the transcription levels of five members increased, including ZmEPF7, 8, 10, 12, and L3.After 24 h under mild water deficit stress, the transcription levels of seven members increased, including ZmEPF6, 7, 9, 11, 12, L2, and L3.Compared with a mild water deficit, more family members responded to a severe water deficit.After 6 h under severe water deficit stress, the transcription levels of seven members increased, including ZmEPF2, 7, 8, 10, 12, L2, and L3.After 24 h under severe water deficit stress, the transcription levels of ten members increased, including ZmEPF2, 6,7,8,9,10,11,12, L2, and L3.
(A) (B) We detected the expression levels of 11 ZmEPF/EPFL family members under drought, heat, and cold treatments by RT-qPCR, including ZmEPF2, 6, 7, 8, 9, 10, 12, 14, 15, L2, and L3 (Figure 8).Results showed that the expression of all these genes was reduced under drought stress.The expression of ZmEPF7, 8, and 15 was induced under heat stress, and ZmEPF7, 10, 12, 14, and L2 were significantly reduced under cold stress (Figure 8).These results suggested that ZmEPF/EPFL members differentially respond to drought, heat, and cold treatments.We detected the expression levels of 11 ZmEPF/EPFL family members under drought, heat, and cold treatments by RT-qPCR, including ZmEPF2, 6, 7, 8, 9, 10, 12, 14, 15, L2, and L3 (Figure 8).Results showed that the expression of all these genes was reduced under drought stress.The expression of ZmEPF7, 8, and 15 was induced under heat stress, and ZmEPF7, 10, 12, 14, and L2 were significantly reduced under cold stress (Figure 8).These results suggested that ZmEPF/EPFL members differentially respond to drought, heat, and cold treatments.In addition, the expression levels of 11 ZmEPF/EPFL family members under different hormone treatments were detected by RT-qPCR (Figure 9), including ZmEPF2, 6, 7, 8, 9, 10, 12, 14, 15, L2, and L3.Results showed that the expression of ZmEPF6, 9, 10, 14, and L3 was significantly reduced, while ZmEPF8 was induced under SA treatment.Under GA treatment, expression of ZmEPF10, 12, 14, L2, and L3 was reduced, while ZmEPF8 was induced.Under ABA treatment, except for ZmEPF7, the expression of ZmEPF8 was obviously induced by ABA, while the expression levels of other members were reduced significantly.Under JA treatment, the expression of ZmEPF10, L2, and L3 was significantly reduced, while ZmEPF8 was induced obviously.Under IAA treatment, the expression of ZmEPF6, 10, 14, L2, and L3 was significantly reduced, while ZmEPF8 was induced obviously.These results suggested that ZmEPF/EPFL members respond to different hormone treatments.In addition, the expression levels of 11 ZmEPF/EPFL family members under different hormone treatments were detected by RT-qPCR (Figure 9), including ZmEPF2, 6, 7, 8, 9, 10, 12, 14, 15, L2, and L3.Results showed that the expression of ZmEPF6, 9, 10, 14, and L3 was significantly reduced, while ZmEPF8 was induced under SA treatment.Under GA treatment, expression of ZmEPF10, 12, 14, L2, and L3 was reduced, while ZmEPF8 was induced.Under ABA treatment, except for ZmEPF7, the expression of ZmEPF8 was obviously induced by ABA, while the expression levels of other members were reduced significantly.Under JA treatment, the expression of ZmEPF10, L2, and L3 was significantly reduced, while ZmEPF8 was induced obviously.Under IAA treatment, the expression of ZmEPF6, 10, 14, L2, and L3 was significantly reduced, while ZmEPF8 was induced obviously.These results suggested that ZmEPF/EPFL members respond to different hormone treatments.

Discussion
Previous studies reported that EPF/EPFL proteins participate in various biological processes in several plant species, including the regulation of stomatal density and aggregation, plant growth, fertility, and yield.However, there is still a lack of systematic identification and comprehensive analyses of the ZmEPF/EPFL family in evolution.In this work, we performed bioinformatics analyses of the EPF/EPFL family members in maize.A total of 18 ZmEPF/EPFL proteins were identified.Their phylogenetic analysis, physicochemical properties, cis-acting element prediction in promoters, and expression pattern analysis were carried out.These results contribute to a better understanding of the evolutionary history and functional mechanisms of the EPF/EPFL family in maize.

Discussion
Previous studies reported that EPF/EPFL proteins participate in various biological processes in several plant species, including the regulation of stomatal density and aggregation, plant growth, fertility, and yield.However, there is still a lack of systematic identification and comprehensive analyses of the ZmEPF/EPFL family in evolution.In this work, we performed bioinformatics analyses of the EPF/EPFL family members in maize.A total of 18 ZmEPF/EPFL proteins were identified.Their phylogenetic analysis, physicochemical properties, cis-acting element prediction in promoters, and expression pattern analysis were carried out.These results contribute to a better understanding of the evolutionary history and functional mechanisms of the EPF/EPFL family in maize.
Phylogenetic tree analysis showed some EPF/EPFL members from monocotyledons (rice and maize) and dicotyledons (Arabidopsis) were distributed in the same branch (Figure 2).For example, ZmEPF3 clusters with AtEPF1 and Os04g0457700 (OsEPF2) and ZmEPF9 and ZmEPF11 clusters with AtEPF2 and Os04g0637300 (OsEPF1), all belonging to Class IV (Figure 2), suggest that the EPF/EPFL family emerged before the differentiation of dicotyledons and monocotyledons.EPF/EPFL members were divided into six subgroups (Figure 2), suggesting that they were highly conserved during the evolution of Arabidopsis, rice, and maize.Moreover, AtEPF1 and AtEPF2 act as negative regulatory factors to regulate stomatal development [10,16], OsEPF1 and OsEPF2 regulate stomatal density [19], and overexpression of OsEPF1 or OsEPF2 significantly reduces the frequency of stomatal density in Arabidopsis epf2 and epf1epf2 mutants, demonstrating that EPF1 and EPF2 are indeed functionally conserved between rice and Arabidopsis.This evidence implies that ZmEPF3, 9, and 11 may function in stomatal development and/or density regulation in maize due to their co-cluster with OsEPF1 and OsEPF2 in Class IV.Similarly, ZmEPFL1, L2, and L3 clustered in Class V with AtEPFL9 (Figure 2), and AtEPFL9 functions as a positive regulatory factor in regulating stomatal density and degree of stomatal aggregation [15,21], suggesting that ZmEPFL1, L2, and L3 also may function in stomatal development in maize.These results indicated that some EPF/EPFL homologies were conserved during evolution.
It was previously reported that the EPF/EPFL gene can appropriately reduce stomatal density in the early stages of plant development, reducing transpiration and water loss while maintaining photosynthesis, thereby improving plant drought resistance and water use efficiency [26], suggesting that the EPF/EPFL gene family may play an important role in defense.Cis-acting element analysis showed that ZmEPF/EPFL members contained defense and stress-responsive elements, low temperature-responsive elements, and drought-inducible elements (Figure 5), speculating that ZmEPF/EPFL members may respond to stress signals.For example, the ZmEPF11 promoter region contains a defense and stress-responsive element (Figure 5), and it is expressed and induced under salt and water deficit, suggesting that it may play a role in response to flooding, salt, UV, and drought (Figure 7).The ZmEPF9 promoter region contains a number of elements, including a defense and stress-responsive element and a drought-inducible element (Figure 5), and it is expressed as induced under water deficit (Figure 7B), suggesting that it may play a role in response to waterlogging and drought.Additionally, RT-qPCR results showed that the expression of ZmEPF7, 8, and 15 was induced under heat stress and ZmEPF7, 10, 12, 14, and L2 were reduced under cold stress (Figure 8).These data indicated that ZmEPF/EPFL members respond to abiotic stresses.
Plant hormones play an important role in the formation and development of stomata [27][28][29][30].For example, auxin (IAA) regulates stomatal development in guard cells.The higher the concentration of auxin, the greater the degree of stomatal opening [31].Several EPF/EPFL members were reported to function in stomatal development and regulation [10,16,21].How these members and hormone signals function in the regulation of stomata together is unknown in maize.Cis-acting element analysis of the promoter region of ZmEPF/EPFL was performed (Figure 5).Results showed that ZmEPF3, 12, and 13 contain auxin-responsive regulatory elements, speculating that these three members may be involved in stomatal development through responding to auxin signals.All ZmEPF/EPFL members contained methyl jasmonate-responsive elements (Figure 5), which is consistent with previous studies on the inhibition of stomatal development by low concentrations of jasmine [32].Additionally, RT-qPCR results showed that the expression of some ZmEPF/EPFL was inhibited or induced under hormone treatments (Figure 9).These data implied that ZmEPF/EPFL members may participate in stomatal development by responding to hormone signals.
In this study, we identified maize EPF/EPFL family members and their potential functions in stomatal development and stress responses.As to what the specific biological functions of each family member are, they need to be analyzed through further in-depth studies.The above exploration results provide a theoretical basis for facilitating further research on the function of ZmEPF/EPFL genes.

Materials
All materials used in the experiment were sourced from the maize B73 inbred line preserved in the laboratory.Plump seeds were selected to be planted in a tissue culture room in a paper cup filled with nutrient soil.The samples were harvested at the three leaf and one heart stages, frozen in liquid nitrogen, and then stored in a −70 • C refrigerator for subsequent experiments.
Seedlings at the three leaves and one heart stage were treated with low temperature (placed in a transparent refrigerator at 4 • C) for 24 h, methyl jasmonate (MeJA, 0.1 mmol/L), abscisic acid (ABA, 0.1 mmol/L), salicylic acid (SA, 0.1 mmol/L), gibberellin (GA, 0.1 mg/L), and auxin (IAA, 0.1 mg/L) for 6 h, respectively.Mock treatment was used as the corresponding control (CK), with three biological replicates for each treatment.Samples from each treatment were harvested for subsequent RT-qPCR analysis.

Distribution of ZmEPF/EPFL Family Members on the Chromosome
Based on the GTF file, the chromosome location information was obtained from gene IDs of the identified maize EPF/EPFL family members.Distribution map on chromosomes of maize EPF/EPFL family members using TBtools-II software [33,34].

Conserved Motif, Domain Prediction, and Phylogenetic Tree Construction of ZmEPF/EPFL Proteins
Conserved motifs of maize EPF/EPFL proteins were predicted using the online MEME (http://meme-suite.org/tools/meme(accessed on 9 January 2024)) software.Conserved domains of maize EPF/EPFL proteins were predicted using CD search tool on the NCBI official website.The conserved motif and domain of maize EPF/EPFL proteins were visualized using the Gene Structure View (Advanced) function of TBtools-II software.All the EPF/EPFL protein sequences of maize, rice, and Arabidopsis were aligned via ClustalW method, and the phylogenetic tree was constructed by MEGA7 (V7.0.26) [36].The phylogenetic tree was further beautified using the iTol online website (https://itol.embl.de/(accessed on 25 January 2024)).

Collinearity Analysis
The genome files and gene annotation files of Zea mays, Oryza sativa, and Arabidopsis thaliana were obtained from the Ensembl Plants database, and the intra-species covariate analysis of Zea mays and inter-species covariate analysis of Zea mays, Oryza sativa, and Arabidopsis thaliana were plotted by the MCScanX function in the TBtools-II software [33,34].

RNA Extraction and Real-Time Fluorescence Quantitative PCR
The total RNAs were extracted from the leaves of each treated plant using an RNAeasy isolation reagent (Vazyme Biotech Co., Ltd., Nanjing, China).The first strands of cDNA were synthesized using the HiFiScript cDNA Synthesis Kit (Vazyme Biotech Co., Ltd.) and then used as the templates of the real-time fluorescence quantitative PCR (qPCR) (Vazyme Biotech Co., Ltd.) for further hormone treatments and cold stress-responsive analysis.The RT-qPCR was performed on the CFX96 real-time PCR instrument (Bio-Rad, Hercules, CA, USA), and ZmActin (GRMZM2G126010) was used as the reference gene.All the primers are listed in Supplementary Table S1.Three biological and technical replicates are performed, respectively.

Figure 1 .
Figure 1.Gene structure and conserved motif prediction of ZmEPF/EPFL family members.(A) The phylogenetic relationship, gene structure, and conserved motifs of cDNA sequences are listed on the picture's left, middle, and right sides, respectively.Gray lines represent introns, green squares represent UTR (untranslated region), and yellow squares represent exons (middle).Gray lines represent proteins expressed by family members; green, red, gray, and yellow boxes represent different motifs (right).(B) The conserved sequences and main protein domains.

Figure 1 .
Figure 1.Gene structure and conserved motif prediction of ZmEPF/EPFL family members.(A) The phylogenetic relationship, gene structure, and conserved motifs of cDNA sequences are listed on the picture's left, middle, and right sides, respectively.Gray lines represent introns, green squares represent UTR (untranslated region), and yellow squares represent exons (middle).Gray lines represent proteins expressed by family members; green, red, gray, and yellow boxes represent different motifs (right).(B) The conserved sequences and main protein domains.

Figure 2 .
Figure 2. Phylogenetic tree of EPFs and EPFLs in Zea mays, Oryza sativa, and Arabidopsis thaliana.Red font indicates EPF/EPFL family members in Zea mays, black font indicates EPF/EPFL family members in Arabidopsis thaliana, and blue font indicates EPF/EPFL family members in Oryza sativa, and different color backgrounds highlight different classes: pink background highlights Class I, blue background highlights Class II, yellow background highlights Class III, purple background highlights Class IV, light yellow background highlights Class V, and green background highlights Class VI.

Figure 2 .
Figure 2. Phylogenetic tree of EPFs and EPFLs in Zea mays, Oryza sativa, and Arabidopsis thaliana.Red font indicates EPF/EPFL family members in Zea mays, black font indicates EPF/EPFL family members in Arabidopsis thaliana, and blue font indicates EPF/EPFL family members in Oryza sativa, and different color backgrounds highlight different classes: pink background highlights Class I, blue background highlights Class II, yellow background highlights Class III, purple background highlights Class IV, light yellow background highlights Class V, and green background highlights Class VI.

Figure 4 .
Figure 4. Gene distribution of ZmEPF/EPFL family members in chromosomes.The black bars represent the chromosomes, the gray lines indicate the location of the genes on the chromosomes, and the length of the chromosomes is shown on the left scale.

Figure 4 .
Figure 4. Gene distribution of ZmEPF/EPFL family members in chromosomes.The black bars represent the chromosomes, the gray lines indicate the location of the genes on the chromosomes, and the length of the chromosomes is shown on the left scale.

Figure 4 .
Figure 4. Gene distribution of ZmEPF/EPFL family members in chromosomes.The black bars represent the chromosomes, the gray lines indicate the location of the genes on the chromosomes, and the length of the chromosomes is shown on the left scale.

Figure 5 .
Figure 5. Cis-element analysis in promoters of ZmEPF/EPFL family members.Gray lines represent promoters corresponding to family members, various colored boxes represent different cis-elements, the cis-element corresponding to each colored box is shown on the right side of the diagram, and promoter lengths are shown on the scale below.

Figure 5 .
Figure 5. Cis-element analysis in promoters of ZmEPF/EPFL family members.Gray lines represent promoters corresponding to family members, various colored boxes represent different cis-elements, the cis-element corresponding to each colored box is shown on the right side of the diagram, and promoter lengths are shown on the scale below.

Figure 6 .
Figure 6.Expression pattern of ZmEPF/EPFL family members in different tissues.The horizontal coordinates represent the tissues from which the material was taken, and the different colors indicate the transcript levels of the genes of each member of the family.

Figure 6 .
Figure 6.Expression pattern of ZmEPF/EPFL family members in different tissues.The horizontal coordinates represent the tissues from which the material was taken, and the different colors indicate the transcript levels of the genes of each member of the family.

Figure 7 .
Figure 7. Expression pattern of ZmEPF/EPFL family members under different stresses.(A) Expression pattern of ZmEPFs/EPFLs under cold, heat, salt, or UV stress.(B) Expression pattern of ZmEPFs/EPFLs under water deficit stress.

Figure 7 .
Figure 7. Expression pattern of ZmEPF/EPFL family members under different stresses.(A) Expression pattern of ZmEPFs/EPFLs under cold, heat, salt, or UV stress.(B) Expression pattern of ZmEPFs/EPFLs under water deficit stress.

Table 1 .
Prediction of physicochemical properties of the maize EPF/EPFL family members.